Project Summary This proposal focuses on the fundamental structural, functional, and mechanistic requirements for the activation of O2 by nonheme iron complexes and related enzymes. Dioxygen is processed by nonheme iron centers in biology as part of a range of critical functions, including the mono- and di-oxygenation of organic substrates, as well as the formation of C-S and C-halide bonds. The oxygenation of organic substrates is mediated by nonheme iron oxygenases, and an important subclass of these enzymes oxygenate sulfur sites bound to the iron center. This subclass includes the thiol dioxygenases (TDOs), such as mammalian cysteine dioxygenase (CDO), and the persulfide dioxygenases (PDOs), such as mammalian ethylmalonic encephalopathy protein (ETHE1). The mechanisms of action of the TDOs and PDOs are poorly understood, although several common iron/oxygen intermediates have been proposed. The sulfoxide synthases EgtB and OvoA are related mononuclear, nonheme Fe enzymes that utilize O2 to carry out both S-oxygenation and C-S bond formation, as does isopenicillin N synthase (IPNS), which employs Fe and O2 in the biosynthetic pathway of penicillin. The C-S bond formation in IPNS occurs via selective carbon radical addition to a sulfur bound to Fe, a process similar to what occurs in nonheme Fe ?-KG halogenases. A number of fundamental mechanistic questions remain unanswered regarding these enzymes. This proposal describes the synthesis and study of synthetic nonheme iron compounds designed to model certain aspects of structure and function related to the TDO/PDOs, sulfoxide synthases, IPNS, and the ?-KG halogenases. Proposed efforts also include select studies on the enzyme CDO, which parallel and complement the model compounds. A focus of the proposal is to characterize reactive, Fe/O2-derived species that are analogs of key intermediates thought to be important in nonheme iron-mediated O2 activation. Characterization of these species in structurally well-defined synthetic complexes will provide precedent and support for the analogous, proposed intermediates in the enzymatic systems. The feasibility of proposed, key bond-making/bond-breaking steps will be established. Methods designed to trap and/or characterize Fe/O2 species will be used, including low temperatures and a suite of advanced spectroscopies (low-temperature UV- vis, electron paramagnetic resonance, resonance Raman, Mssbauer, X-ray absorption). Computational studies will be employed to help interpret and predict structural and spectroscopic properties as well as reaction pathways. The selective reactivity of carbon radicals with iron-heteroatom bonds will also be assessed, taking advantage of a unique set of new, structurally characterized ferric hydroxide complexes. These studies should lead to significant advances in our fundamental knowledge regarding how nonheme Fe enzymes activate O2 and selectively oxidize substrates. This knowledge should also provide guidance for the design of future transition metal catalysts. The misfunctioning of these enzymes have been implicated in a variety of diseases, including neurodegenerative disorders (Alzheimer's, Parkinson's), arthritis, cancer, and genetic disorders.